Over the last year, we have investigated a possible impact of genetic diversity on the distribution and sero-reactivity of linear antigenic epitopes in gp120 encoded in new African HIV-1 subtypes A, C, and D, and in new North American/European subtype B. A region of the ENV gene that encodes C2 to the N-terminus of gp41 was amplified from primary peripheral blood leukocytes or from short-term cultured isolates. The amplified DNA was cloned, sequenced and translated. The amino acid residues were analyzed for distribution and immuno- reactivity of the antigenic sites, predicted using protein surface probability parameters. Despite variable levels of diversity in the primary sequence, eight analogous epitopes of high antigenic potential, including E2 (300-320), E3 (335-344), E4 (351-355), E7 (358-385), and E8 (431437), were identified in the virus from all clades. Analysis of the amino acid sequence revealed that E2 corresponds to the PND previously identified in the V3 loop. Synthetic peptides comprising the epitopes were tested for reactivity with the serum samples from asymptomatic African donors, Thai donors, and anti-gp120 antibodies developed in mice using plasmid DNA inoculation. Each of the epitopes reacted at various intensities with the patients' sera. The reactivity of gp120 in ELISA was attributable to E2 (50%) >E7 (21%) >E8 (13%) >E4 (8%) = E3 (8%). The E2 epitope showed a marked degree of variability whereas E3, E7, and E8 showed more uniform patterns of reactivity with the antibodies of the indigenous African subjects. Some of the residues comprising E7 have been previously mapped to the conserved neutralizing determinant associated with the CD4 binding region. The Thai serum samples showed negligible reactivity with the epitopes derived from group A or group D subtypes. UG23c, a prototypic group D virus which lacks a B-turn conformation in the V3 loop, exhibited a markedly reduced antigenic index at the E2 site (PND), and was largely unreactive with most of the indigenous test sera. The anti-gpl2O antibodies developed in mice reacted with all major epitopes previously implicated in virus neutralization. A strong correlation was observed in the ability of the mouse and human serum antibodies to react with these major antigenic sites. DNA inoculation appears to elicit antibodies that recognize both variable and conserved neutralizing epitopes expressed by the virus in a natural infection. The findings further demonstrates the efficacious application of synthetic peptides to define the antigenic relationships among divergent clades of HIV.